We intend to define the mechanism(s) determining the specificity of mutation induced by agents which block DNA synthesis. We propose to test the hypothesis that such mutagens produce non-instructional lesions and that the specificity of mutation at non-instructional lesions is determined primarily by the properties of the replicative system rather than by the base pairing properties of the altered base(s). In order to accomplish these goals we will continue studies with an in vitro model system in which a small viral DNA template strand of known sequence is reacted with mutagen. In vitro synthesis on such templates ordinarily terminates just before the damaged nucleotide but under certain experimental conditions, nucleotides can be inserted opposite the lesion. We have already determined, with three different mutagens, that there is a preference for the insertion of A(denine) opposite non-instructional sites. If this is a universal rule, it suggests some general predictions about the specificity of mutational change. Treatments which inactivate pyrimidines, producing non-instructive lesions should produce an excess of transitions. Inactivation of purines, resulting in non-instructive lesions should produce an excess of transversions, particularly (for Guanine) the G:C to T:A transversion. We propose to test the generality of our observations: a) by the use of other mutagens to increase the data base; b) by development of a system in which double stranded DNA is replicated; c) by the development of an in vitro/in vivo system with which we can examine genetic as well as biochemical events; and d) by applying the results to replicating systems in vivo. Our results, so far, pertain to base substitution mutagenesis. The in vitro results suggest that we can also use our approach to study the events in frame-shift mutagenesis. Recent data from other laboratories emphasizes the importance of mutational change in carcinogenesis. We expect that the proposed studies will provide information on the mechanism of such mutational change.